Only small quantities of certain peptides, including human amylin, are available from procedures which involve natural isolation. Amylin is a 37 amino acid peptide hormone which was recently discovered, isolated and purified by Cooper and Willis. European Patent Application No. 88303803.6 ("Amyloid Peptides"). Cooper also determined that amylin has marked effects on carbohydrate metabolism. See, e.g., Cooper et al., Proc. Nat. Acad. Sci. USA 84:8628-8632 (1987). Various patent applications relating to uses of amylin, amylin agonists, and amylin antagonists for the treatment of certain disorders, such as diabetes, have been prepared. European Patent Application No. 88307927.9 ("Use of Amylin or CGRP for the Treatment of Diabetes Mellitus"); International Application No. PCT/US89/00049 ("Treatment of Type 2 Diabetes Mellitus"). Commonly assigned and co-pending patent application U.S. Ser. No. 667,040 is directed to the synthesis of this amylin using a solid phase resin synthetic method. The application describes the synthesis of this hormone using solid phase methods which makes it available in greater quantities for extensive research, including anticipated clinical trials.
However, even conservative estimates as to the commercial requirements of amylin lead to the conclusion that about 50 to 500 kg will be needed annually. The largest protein to date made commercially by solid phase resin synthesis is salmon calcitonin which has 32 amino acids and which is synthesized on a scale of about 10 kg/year. The labors of synthesis increase geometrically with increased amino acid chain length; thus, synthesis of a 37 amino acid peptide is more formidable than a 32 amino acid peptide. Also the particular amino acid residue content of amylin may increase the burdens of synthesis using solid-phase methods. For the above reasons, options other than resin synthesis may be of value for larger scale synthesis of more complex proteins like amylin. As the requirements for larger and larger amounts of end product grow, equipment limitations and costs of amino acids and other reagents, along with waste disposal can make such synthesis procedures technically difficult and prohibitively expensive.
Recombinant DNA techniques may provide an attractive approach to the synthesis of amylin in commercial amounts. Some large proteins are now made commercially by this methodology (e.g., .alpha.-interferon, interleukin-2, tissue plasminogen activator, Factor VIII:C, erythropoietin). However, high levels of expression of these proteins by such biological systems are required to make their manufacture, isolation and purification commercially feasible. E. coli and yeast expression systems are capable of providing high yields of proteins by recombinant technology; however, these systems are not capable of performing certain post-translation modifications. For example, they do not construct or express peptide amides, only peptide acids. Amylin is a peptide amide that is much less biologically active in its peptide acid form. A few recombinant expression systems have been reported to provide the amide form of a precursor peptide acid; however, the recombinant expression systems that do provide the amide form of proteins, such as mammalian cells and the baculovirus expression vector system, do so in low yields. These yields would be too low to efficiently and economically provide the commercial quantities of amylin that would be required.
Several methods of enzymatic transformation of "protein precursor acids" to give a peptide amide have been described. Use of an .alpha.-amidating enzyme system isolated from rat medullary thyroid carcinomas to prepare an .alpha.-amidated polypeptide from a polypeptide substrate having a C-terminal glycine residue has been reported by one group. See Beaudry, G. A. et al., Journal of Biological Chemistry 265:(29):17694-17699 (1990); and U.S. Pat. No. 4,708,934. The amide function is reportedly donated to the polypeptide by cleavage from the .alpha.-amino group of the terminal glycine residue of a precursor polypeptide acid. The resulting .alpha.-amidated peptide has one less amino acid residue, the glycine residue having been eliminated.
This enzymatic amidation has proved difficult to reproduce and yields of product decrease exponentially as the size of the protein to be amidated is increased. The cost of the enzyme by natural extraction renders this process economically unacceptable on commercial scale, and the .alpha.-amidating enzyme has now been cloned, expressed, synthesized, and isolated by recombinant techniques. However, the use of the recombinantly synthesized enzyme on a precursor polypeptide having a C-terminal glycine acid added to the sequence which itself has been made by recombinant technologies would make the resulting process still unacceptably expensive for very large scale production of amylin (i.e., in the 500 kilogram per year synthesis scale).
Other enzymatic transformations reported to give protein amides involve proteases. One such method was said to convert small peptides to peptide amides using carboxypeptidase II. Klaus Breddam, Carlsberg, Res. Comm. Vol 50, p. 209 (1985). Another method was reported to generate peptides having a C-terminal amide using carboxypeptidase Y and small peptides, and in one case, human calcitonin-Leu. Most yields with this method were reported to be less than 25% (see U.S. Pat. No. 4,709,014, assigned to Sankyo Company Limited), and yields of peptide amides using such enzyme systems have been reported to decrease as the complexity or length of the peptide chain increases.
Aside from amylin, other peptide amides having biological activity include thyrotropin releasing hormone (TRH), oxytocin, vasopressin, luteinizing hormone releasing hormone, melanocyte stimulating hormone (MSH), gastrin, CGRP-1, CGRP-2, Substance P, secretin, the calcitonins, growth hormone releasing hormone, and vasoactive intestinal peptide (VIP).
Thus, it is important to provide an economically attractive and technically simple route to transform protein acids to amides. Unfortunately, while simple chemical transformations of carboxylic acids to carboxamides are known, the reagents usually involved in such reactions may destroy the sensitive protein backbone. In addition, as the complexity of the peptide sequence and structure increases, competing reactions with other reactive groups in the amino acid side chains increase, and such side reactions may drastically reduce the yield of the desired peptide amide.